Summary: The long-term goals of this project are: 1) to evaluate the feasibility of generating live attenuated virus vaccines of HIV-1 and HIV-2 that are rendered non-pathogenic by mutation of accessory genes, either individually or in combination; 2) to explore the possibility that the accessory gene proteins can be targets for anti-viral therapy; and 3) to generate retroviruses that replicate without integrating into the host genome. As prerequisites to the development of candidate live attenuated virus vaccines and the development of anti-HIV drugs directed against the accessory gene products, we have been engaged on studies to determine the role of these proteins in the life cycle of HIV-1 and HIV-2 in vitro, since a knowledge of how they function is critical to both goals. Our earlier work had demonstrated the critical role of HIV-1 Vif to virus replication in primary T cells (peripheral blood mononuclear cells, PBMC) and in primary monocyte-derived macrophages (MDM). In the case of Nef, we have shown that whether or not Nef has a measurable effect on virus replication depends on the particular virus-host system used. While Nef mutants of several HIV-1 strains all replicate slightly less well than wild type in PBMC and in MDM, there can be either no effect or dramatic reductions in virus replication when Nef mutants of HIV-1 and HIV-2 are assayed in CD4-positive cell lines. As part of our goal to develop attenuated HIV vaccine candidates, we have modified an HIV-1 genome to allow the insertion of different genes into the nef open reading frame. The vif genes of HIV-1 and HIV-2 have been inserted into the both the homologous and heterologous viruses with the aim of determining whether ectopic expression of Vif is functional and whether Vif of HIV-1 can complement Vif mutants of HIV-2 and vice versa. Preliminary work has demonstrated that HIV-2 vif can complement HIV-1 Vif mutants, but that vif from SIVagm cannot. This work will be extended to a study of Nef and Vpr. The group has recently become interested in the vpu gene of HIV-1 because of the fact that several clones of HIV-1 have been found to have a mutated vpu gene and Vpu has been shown to enhance infectivity in MDM. For example, both the AD and the MAL clones had mutations that resulted in a Vpu-minus virus. Correction of the vpu mutation in AD enhanced its replication in MDM. MAL is one of the few examples of an R5 virus that cannot establish a productive infection in MDM despite the fact that the MAL Env can mediate entry into MDM and MDDC. The group is trying to map the determinants of macrophage tropism and are using MAL as an example of an R5 virus that is not M tropic. Determining whether a Vpu-plus version of MAL is now able to replicate in MDM is one of the steps. If this is not sufficient, then hybrid viruses will be constructed between an M-tropic virus (e.g., AD) and MAL.